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Markedly reduced axonal potassium channel expression in human sporadic amyotrophic lateral sclerosis: An immunohistochemical study
Experimental Neurology 232 (2011) 149–153
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Markedly reduced axonal potassium channel expression in human sporadic amyotrophic lateral sclerosis: An immunohistochemical study Kazumoto Shibuya a, Sonoko Misawa a, Kimihito Arai b, Miho Nakata a, Kazuaki Kanai a, Yasumasa Yoshiyama b, Kimiko Ito b, Sagiri Isose a, Yu-ichi Noto a, Saiko Nasu a, Yukari Sekiguchi a, Yumi Fujimaki a, Shigeki Ohmori a, Hiroshi Kitamura c, Yasunori Sato d, Satoshi Kuwabara a,⁎ a
Department of Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Neurology, Chiba East National Hospital, Chiba, Japan c Clinical Research Center, Chiba East National Hospital, Chiba, Japan d Clinical Research Center, Chiba University Hospital, Chiba, Japan b
a r t i c l e
i n f o
Article history: Received 27 June 2011 Revised 27 July 2011 Accepted 18 August 2011 Available online 30 August 2011 Keywords: Amyotrophic lateral sclerosis Potassium channel Motor axon Axonal hyperexcitability Fasciculation
a b s t r a c t Fasciculations are characteristic features of amyotrophic lateral sclerosis (ALS), suggesting abnormally increased excitability of motor axons. Previous nerve excitability studies have shown reduced axonal potassium currents in ALS patients that may contribute to the hyperexcitability and thereby generation of fasciculations. To clarify changes in axonal ion channel expression in motor axons of ALS, we performed immunohistochemistry of potassium and sodium channels in the C7 and L5 ventral/dorsal roots obtained from five autopsy cases of sporadic ALS. Compared to controls, the immunoreactivity of potassium channels (Kv1.2) was markedly reduced in the ventral roots, but normal in the dorsal roots of all the ALS patients. Nodal sodium channel expression was not significantly different in ALS patients and control subjects. Our results show prominently reduced expression of axonal potassium channels, and provide the neuropathological and biological basis for decreased accommodative potassium currents in motor axons of ALS patients. The axonal hyperexcitability would lead to generation of fasciculations, and possibly enhances motor neuron death in ALS. © 2011 Elsevier Inc. All rights reserved.
Introduction Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disorder characterized by upper and lower motor neuron loss with fasciculations (Cleveland and Rothstein, 2001; Ringel et al., 1993). Extensive fasciculations are prominent features of ALS, presumably mediated by increased excitability of some part of the motor neuronal membrane (de Carvalho, 2000; Layzer, 1994). Fasciculations may occur before any motor neuron death, as a precursor to ALS (de Carvalho and Swash, 1998). A better understanding of their membrane mechanism may therefore provide insights into the patholphysiology of ALS. Although the mechanisms underlying the selective death of motor neurons remain poorly understood, there are several hypotheses, such as oxidative stress, protein aggregation, neuroinflammation, growth factor deficiency, impaired axonal transport, and excitotoxicity (Van Damme et al., 2005). Of these, excitotoxicity would lead to abnormal neuronal hyperexcitability of motor neurons through increased calcium ion influx into motor neuronal cell body resulting in frequent fasciculations (Brown, 1994; Henneberry et al., 1989; Novelli et al., 1988; Van Den Bosch et al., 2006). However, there are several lines of evidence ⁎ Corresponding author at: Department of Neurology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. Fax: + 81 43 226 2160. E-mail address: [email protected] (S. Kuwabara). 0014-4886/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2011.08.015
that the predominant anatomical sites of origin of fasciculations are the distal motor axons (Layzer, 1994), although some fasciculations arise proximally early in the disease (de Carvalho and Swash, 1998). Therefore, most fasciculations arise from axons rather than neuronal cell bodies, suggesting altered membrane properties in motor axons of ALS. Previous axonal excitability studies in ALS patients have shown two types of ion channel abnormalities, reduced fast potassium currents and increased persistent sodium currents, both of which lead to axonal hyperexcitability (Bostock et al., 1995; Kanai et al., 2006; Mogyoros et al., 1998; Vucic and Kiernan, 2006). However, excitability indices measured by the neurophysiological technique are indirect measures of axonal ion channel function, and so far direct morphological examinations of altered ion channel expression have never been performed. By using immunohistochemistry, we studied the changes in expression of the ion channels in motor axons obtained from five autopsy cases of sporadic ALS. Materials and methods ALS patients and control subjects We studied post-mortem materials obtained from five patients (3 men and 2 women) with pathologically-proven ALS. Clinical profiles are shown in Table 1. The median survival time was 33 months (range, 22 to 49 months). None of them had a family history of ALS.
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Table 1 Clinical profiles of ALS patients and control subjects. Subject
Age at death gender
Site of onset
Survival time (months)
Postmortem delay (h)
Bulbar Bulbar Upper limb Upper limb Upper limb
49 22 25 27 42
2 5 4 6 2
96 84
4 12
Patient 1 2 3 4 5
ALS ALS ALS ALS ALS
64 73 64 70 68
Control 1 2
LE AD
57 F 84 M
M F M F M
ALS= amyotrophic lateral sclerosis; LE= leukoencepharopathy; AD= Alzheimer's disease.
TDP-43 immunostaining in the cytoplasm of spinal motor neurons was positive in all five patients. Two patients with Alzheimer disease or leukoencephalopathy with spheroids served as disease controls (Freeman et al., 2009). The research protocol was approved by the local Research Ethics Committees. Informed consent of immunohistological investigations was obtained their family members. Immunohistochemistry In all subjects, the hypoglossal nerves, and C7 and L5 ventral/dorsal roots were collected 2–12 h after death (Table 1). C7 and L5 roots were chosen because of the anatomical reason; these roots are located in the center of the cervical (C5–T1) and lumbar (L3–S1) enlargements of the spinal cord. Tissues were post-fixed for 30 min in 4% paraformaldehyde in 0.1 M PB, pH 7.4. And these were cryoprotected with 30% sucrose in phosphate-buffered saline (PBS), pH 7.4, overnight at 4 °C, as described previously (Ishibashi et al., 2004). After flat embedding in rectangular moulds in OCT medium, nerves were sectioned longitudinally at 8 μm. Cryosections were permeabilized for 1 h in 0.1 M PB, pH7.4, containing 0.3% Triton X-100 and 10% goat serum (PBTGS). For double-labeling experiments, sections were incubated overnight at 4 °C with primary antibodies diluted to appropriate concentrations in PBTGS. Primary antibodies were a rabbit polyclonal antibody against Kv1.2 channel (1:200) and mouse monoclonal antibody against pan sodium channel (1:500). These were purchased from Alomone labs (Jerusalem, Israel) and Sigma Co. (St. Louis, Missouri, USA). The sections were thoroughly rinsed in PBS, followed by application of fluorescently labeled secondary antibodies used were Alexa Fluor 488-labeled goat anti-rabbit immunoglobulin G (IgG) and Alexa Fluor 594-labeled goat anti-mouse IgG (1:2000; Molecular Probes, Eugene, Oregon, USA). Analysis of tissue sections For analysis of nerve sections, multiple images were captured with confocal microscope FV10i (Olympus Co Ltd, Tokyo, Japan). In each nerve, 30 fields were randomly captured and each fieldconsisted of 117.85 × 117.85 μm. To ensure random sampling, 10 fields were selected for analysis as performed in previous studies (Craner et al., 2003). Sensitivities to the both fluorescence were fixed for capturing each images. Signal intensities of the immunostaining signal at sodium and potassium channel cluster was obtained by manual outlining of each channels, using Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA) integrated densitometry function to calculate mean signal intensities for the outline area. (0–255) Background signal was subtracted from both ALS and control images (Craner et al., 2003). TDP-43 histochemistry In post-mortem materials from 5 ALS patients, sections (10 mm) of formalin-fixed, paraffin-embedded spinal cord were examined. Sections
were immunostained with anti phosphorylated TDP-43 antibodies (mouse monoclonal, 1:5000; rabbit polyclonal, 1:5000; Cosmobio, Tokyo, Japan).
Multiple Excitability Measurements Multiple excitability properties were measured for the median nerve at the wrist in Patient 5, (QTRAC version 4.3 with multiple excitability protocol TRONDXM2; copyright, Prof. Hugh Bostock, Institute of Neurology, London, UK), as reported elsewhere (Kanai et al., 2006; Kiernan et al., 2000). Briefly, in the threshold electrotonus studies, the membrane potential was altered by use of DC polarizing currents that were 40% of the unconditioned threshold. Depolarizing and hyperpolarizing currents were used, each lasting 100 ms, and their effects on the threshold current for the test motor responses were examined. The recovery cycle of axonal excitability after a single supramaximal stimulus was measured by delivering the test stimulus at different intervals after the conditioning stimulus. The interval between the conditioning and test stimulation were changed systematically from 2 to 200 ms.
Statistical analyses A repeated measurement analysis (Fitzmaurice et al., 2004) was performed to assess the immunostaining intensity of potassium and sodium channel clusters. F-test was used for adjustment of comparison between controls and ALS patients. Dunnett's multiple comparison tests were also applied between controls and ALS subgroups. In all comparisons, p-value of less than 0.05 was considered to be statistically significant. All statistical analyses were performed by the SAS software program, version 9.2 (SAS Institute Inc., Cary, NC, USA).
Results Immunostaining of potassium channel clusters In ALS patients, the immunoreactivity of potassium channel clusters (Kv1.2 channels) was prominently reduced in the ventral roots at both the C7 and L5 levels. Fig. 1 shows examples of immunostaining in the C7 roots of Patient 3 and a control subject with Alzheimer disease. In the ALS patient, the immunoreactivity of Kv1.2 channels was nearly lost in the C7 ventral root (Fig. 1C), but the potassium channel clusters were similarly stained in the ALS dorsal root and control ventral/dorsal roots. Table 2 shows results of the immunostaining intensity measured by densitometry for all subjects. For potassium channel clusters, the mean intensities markedly decreased in the C7 and L5 ventral roots in the ALS group than in the controls. In the hypoglossal nerves, the intensities were reduced in patients with bulbar onset (patients 1 and 2), but the difference did not reach a significance because of severe loss of axons and thereby a small number of nodes examined. Patient 3 with upper limb onset showed the decreased intensities predominantly in the C7 ventral root with relatively preserved those in the L5 ventral root (Fig. 2). ALS patients with lower limb onset (patients 4 and 5) had prominently reduced intensities in both the C7 and L5 ventral roots, whereas the extent of reduction was greater in the L5 roots. The immunostaining intensities in the C7 and L5 dorsal roots were similar for ALS patients and control subjects.
Immunostaining intensity of sodium channel clusters Table 2 also shows the immunostaining intensities of nodal sodium channel clusters. The intensities were not significantly different in the hypoglossal nerve, C7 ventral and dorsal roots, and L5 ventral and dorsal roots of control subjects and ALS patients.
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Discussion Our results show that in ALS patients, expression of Kv1.2 channels is markedly reduced in motor axons, and normal in sensory axons in both the C7 and L5 spinal roots levels, indicating selective loss of potassium channels in motor axons of ALS patients. These findings provide the morphological and biological basis for the previous neurophysiological data of reduced accommodative axonal potassium currents, and resulting increased axonal excitability in ALS motor axons (Bostock et al., 1995; Kanai et al., 2006; Vucic and Kiernan, 2006).
Prominently reduced expression of potassium channel in the ventral roots
Fig. 1. Immunohistochemistry of sodium and potassium channels in a patient with ALS and control subject. Longitudinal sections of the C7 ventral/dorsal roots double immunostained for pan-Nav channel (red) and Kv1.2 (green) in a post-mortem specimen obtained from autopsy cases of Alzheimer disease (A, B) and amyotrophic lateral sclerosis (C, D) (ALS; Patient 3 in Table 1). Note marked loss of the immunoreactivity for Kv1.2 channels in ALS ventral root (C). A scale bar indicates 10 μm.
Axonal excitability studies Patient 5 underwent axonal excitability studies in median motor axons at the wrist 22 months before death. Fig. 3 shows threshold electrotonus and the recovery cycle of axonal excitability after a single supramaximal conditioning stimulus. Threshold electrotonus demonstrated greater threshold changes in the early depolarizing phase (less accommodative currents mediated by fast potassium channels), and the recovery cycle showed increased superexcitability (less fast potassium currents). These findings suggested reduced fast potassium currents (Kv1.2 channels) as reported in previous studies (Bostock et al., 1995; Kanai et al., 2006; Vucic and Kiernan, 2006).
Our findings are highly consistent with previous neurophysiological study results. Axonal excitability studies in ALS patients have shown decreased fast and slow potassium currents in motor axons of the median or ulnar nerve at the wrist (Bostock et al., 1995; Kanai et al., 2006; Vucic and Kiernan, 2006). The functional reduction of potassium currents is now proved to be caused by the decreased channel expression. A microarray analysis in the spinal motor neurons of ALS patients has shown reduced mRNA expression of several types of potassium channels (KCNA1, KCNA2 and KCNQ2) (Jiang et al., 2005). Kv1.1 is a shaker related voltage-gated potassium channel in humans encoded by the KCNA1 gene, Kv1.2 channel examined in this study is encoded by the KCNA2 gene, and Kv7.1 channel is encoded by the KCNQ2 gene. Combined with the microarray study results, our findings suggest that the reduced mRNA levels of potassium channels in the spinal motor neurons lead to less expression of potassium channel proteins in peripheral motor axons in ALS. In addition, previous studies in transgenic mice revealed that axonal transport deficiencies could play important roles in the pathophysiology of ALS, and impaired transport of axonal membrane proteins including several potassium channels or proteins required for maintaining potassium channels might also contribute to a reduction of expression in potassium channels (LaMonte et al., 2002). Overall, the combination of decreased potassium channel mRNA and impaired axonal transport could result in markedly reduced axonal potassium channel expression, and thereby decreased potassium currents. Because potassium currents are compensatory outward rectifying conductances in response to depolarization, their decrease would results in substantial increases in axonal excitability and spontaneous firing of motor axons (e.g., fasciculations). It is recently suggested that a subset of ALS patients may have serum anti-potassium channel antibodies (Nwosu et al., 2010; Thompson and
Table 2 The immunostaining intensity of potassium and sodium channel clusters. Control (n = 2) LS mean (95%CI)
Amyotrophic lateral sclerosis All (n = 5)
Bulbar (n = 2)
Upper limb (n = 1)
Lower limb (n = 2)
LS mean (95%CI)
P-valuea
LS mean (95%CI)
P-valueb
LS mean (95%CI)
P-valueb
LS mean (95%CI)
P-valueb
Potassium channel cluster Hypoglossal nerve C7 ventral root C7 dorsal root L5 ventral root L5 dorsal root
28.4 45.6 42.2 47.1 46.6
(− 45.8–102) (32.6–58.5) (29.3–55.0) (30.7–63.4) (36.8–56.3)
18.9 18.7 34.8 24.1 42.1
(− 0.71–38.5) (9.27–28.2) (29.7–40.0) (13.6–34.6) (37.1–47.1)
0.55 0.0061 0.10 0.024 0.30
5.77 23.7 37.4 42.7 44.4
(− 74.3–85.8) (1.39–46.0) (21.9–52.9) (21.5–63.9) (39.5–49.2)
0.73 0.16 0.81 0.92 0.84
21.7 24.0 33.9 41.6 36.1
(− 55.4–98.9) (3.95–44.1) (19.0–48.8) (17.9–65.4) (23.5–48.8)
0.98 0.14 0.51 0.88 0.24
21.6 12.6 34.3 9.73 42.8
(− 32.8–76.1) (− 3.73–29.0) (20.5–48.1) (− 6.26–25.7) (33.1–52.6)
0.97 0.034 0.51 0.030 0.73
Sodium channel cluster Hypoglossal nerve C7 ventral root C7 dorsal root L5 ventral root L5 dorsal root
34.7 48.7 52.6 54.3 58.9
(2.39–67.0) (6.40–91.0) (15.9–89.3) (− 2.11–110) (29.9–87.9)
31.9 46.2 45.6 68.0 57.3
(22.6–41.3) (25.0–64.4) (30.8–60.5) (55.6–80.3) (45.0–69.5)
0.70 0.87 0.54 0.37 0.86
31.5 40.8 43.5 74.9 60.1
(− 7.34–70.4) (− 4.27–85.9) (6.38–80.8) (16.8–133) (31.2–88.9)
0.98 0.95 0.90 0.78 0.99
36.3 66.5 44.8 74.6 50.6
(2.12–70.5) (6.41–126) (− 6.70–96.3) (− 5.39 -154) (9.67–91.5)
0.99 0.80 0.95 0.85 0.91
29.9 40.5 48.1 58.3 57.7
(5.83–54.1) (− 2.50–83.6) (11.2–85.0) (1.74–114) (28.5–86.9)
0.91 0.94 0.98 0.99 0.99
LS Mean: least square mean. a F test P-value by repeated ANOVA. b Adjusted P-value by multiple tests using Dunnett's test.
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Fig. 2. Immunostaining reactivity measured by densitometry. The immunostaining reactivity of potassium channel (Kv1.2 channel) cluster measured by densitometry in the C7 and L5 ventral/dorsal roots obtained from autopsy cases of amyotrophic lateral sclerosis (ALS; n = 5) and controls (n = 2). Note prominent reduction of the intensities in the ALS ventral roots. In the dorsal roots, the immunostaining intensity was preserved in ALS patients. B = Bulbar onset (n = 2); U = upper limb onset (n = 1); L = lower limb onset (n = 2). *p b 0.05; **p b 0.001.
Gan, 2011). We did not measure the antibodies in our patients, but this issue should be clarified in future studies. In this study, immunostaining intensities of potassium channel clusters in sensory axons were preserved in ALS. Clinically and electrophysiologically, sensory axons are preserved in ALS (de Carvalho, 2000; Ertekin, 1967; Fincham and Vanallen, 1964), and excitability of sensory axons is not altered in ALS (Mogyoros et al., 1998). It is generally accepted that sensory axons are not involved in ALS, and our results indicate altered expression of potassium channels is selective for motor axons in ALS. Distribution of the reduced potassium channels and clinical phenotypes Our results show a tendency that the immunostaining intensities of potassium channel cluster were more severely decreased in the spinal roots corresponding to the site of symptom onset than other segments (Table 2, Fig. 2); ALS patients with lower limb onset tended to show the lower immunostaining intensity in the lumbar roots. These findings appear to be consistent with a pattern of progression in the ALS pathology (Brooks, 1996; Caroscio et al., 1987). Lower motor neuron degeneration in ALS is a focal process that advances contiguously (Ravits et al., 2007). Reduction of potassium currents leads to hyperexcitability in motor neurons/axons, and motoneuron may no longer meet the demands of its ATP-dependent processes, and ultimately dies (Henneberry et al., 1989; Novelli et al., 1988; Van Den Bosch et al., 2006). Conversely, the predominancy of potassium channel changes may determine the site of onset in ALS. Preserved immunostaining intensities of nodal sodium channels Our findings also show that there is no significant change in sodium channel expression in motor axon of ALS patients. Previous excitability studies revealed an increase in persistent sodium current in motor axon with ALS (Bostock et al., 1995; Kanai et al., 2006;
Mogyoros et al., 1998; Vucic and Kiernan, 2006). Sodium channels in the nodes of Ranvier of the peripheral nerves consist of only Nav1.6 channels, but there are two functionally different channels among Nav1.6 channels; in normal mammalian myelinated nerves, 1.0%–2.5% of the total nodal sodium channels are open at rest, and therefore termed “persistent” channels (Mogyoros et al., 1998). Our study failed to confirm changes in sodium channels. Immunohistochemistry cannot differentiate classical transient sodium channels and persistent channels, because these channels are structurally the same. Therefore normal sodium channel expression in this study does not exclude changes in persistent sodium currents. Another possible reason for the lack of changes in sodium channels in ALS includes the disease stage at the time of examination. Our previous study has shown that persistent sodium currents estimated by strength-duration time constant were increased in the whole ALS group, but were not significantly different in the end stage when patients develop severe muscle wasting (Kanai et al., 2006). In this regard, our findings were consistent with previous studies that suggest increase persistent sodium channels in the whole ALS patients examined (Mogyoros et al., 1998). Relevance to treatment Our morphological study and previous physiological studies revealed reduction of potassium channel expression and resulting decreased potassium currents in motor axon with ALS (Bostock et al., 1995; Kanai et al., 2006; Mogyoros et al., 1998). Investigating ionic mechanisms is of clinical relevance, because once a specific ion channel abnormality is identified, its pharmacologic modulation would provide a new therapeutic option. Riluzole is the only therapeutic agent for which effectiveness in human ALS patients has been confirmed by a large randomized placebo-controlled trial (Lacomblez et al., 1996). The positive effects of riluzole are related with its anti-excitotoxic properties inhibition of
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References
Fig. 3. Excitability testing in Patient 1 before death. Multiple axonal excitability testing for the median nerve at the wrist in a patient with amyotrophic lateral sclerosis (ALS; Patient. 5 in Table 1) 22 months before death. Thick lines indicate data from an ALS patient, and thin lines, the mean curves of age-matched normal subjects (n = 10). A. Threshold electrotonus showed greater threshold changes in the early depolarizing phase (arrow). B. Recovery cycle of axonal excitability after a single supramaximal conditioning stimulus showed greater supernormality in an ALS patient (arrow). These finding suggest reduced fast potassium currents mediated by Kv1.2. Strength-duration time constant was 0.43 ms (normal mean + − SEM; 0.43 + − 0.02 ms; n = 10).
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Markedly reduced axonal potassium channel expression in human sporadic amyotrophic lateral sclerosis: An immunohistochemical study Kazumoto Shibuya a, Sonoko Misawa a, Kimihito Arai b, Miho Nakata a, Kazuaki Kanai a, Yasumasa Yoshiyama b, Kimiko Ito b, Sagiri Isose a, Yu-ichi Noto a, Saiko Nasu a, Yukari Sekiguchi a, Yumi Fujimaki a, Shigeki Ohmori a, Hiroshi Kitamura c, Yasunori Sato d, Satoshi Kuwabara a,⁎ a
Department of Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Neurology, Chiba East National Hospital, Chiba, Japan c Clinical Research Center, Chiba East National Hospital, Chiba, Japan d Clinical Research Center, Chiba University Hospital, Chiba, Japan b
a r t i c l e
i n f o
Article history: Received 27 June 2011 Revised 27 July 2011 Accepted 18 August 2011 Available online 30 August 2011 Keywords: Amyotrophic lateral sclerosis Potassium channel Motor axon Axonal hyperexcitability Fasciculation
a b s t r a c t Fasciculations are characteristic features of amyotrophic lateral sclerosis (ALS), suggesting abnormally increased excitability of motor axons. Previous nerve excitability studies have shown reduced axonal potassium currents in ALS patients that may contribute to the hyperexcitability and thereby generation of fasciculations. To clarify changes in axonal ion channel expression in motor axons of ALS, we performed immunohistochemistry of potassium and sodium channels in the C7 and L5 ventral/dorsal roots obtained from five autopsy cases of sporadic ALS. Compared to controls, the immunoreactivity of potassium channels (Kv1.2) was markedly reduced in the ventral roots, but normal in the dorsal roots of all the ALS patients. Nodal sodium channel expression was not significantly different in ALS patients and control subjects. Our results show prominently reduced expression of axonal potassium channels, and provide the neuropathological and biological basis for decreased accommodative potassium currents in motor axons of ALS patients. The axonal hyperexcitability would lead to generation of fasciculations, and possibly enhances motor neuron death in ALS. © 2011 Elsevier Inc. All rights reserved.
Introduction Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disorder characterized by upper and lower motor neuron loss with fasciculations (Cleveland and Rothstein, 2001; Ringel et al., 1993). Extensive fasciculations are prominent features of ALS, presumably mediated by increased excitability of some part of the motor neuronal membrane (de Carvalho, 2000; Layzer, 1994). Fasciculations may occur before any motor neuron death, as a precursor to ALS (de Carvalho and Swash, 1998). A better understanding of their membrane mechanism may therefore provide insights into the patholphysiology of ALS. Although the mechanisms underlying the selective death of motor neurons remain poorly understood, there are several hypotheses, such as oxidative stress, protein aggregation, neuroinflammation, growth factor deficiency, impaired axonal transport, and excitotoxicity (Van Damme et al., 2005). Of these, excitotoxicity would lead to abnormal neuronal hyperexcitability of motor neurons through increased calcium ion influx into motor neuronal cell body resulting in frequent fasciculations (Brown, 1994; Henneberry et al., 1989; Novelli et al., 1988; Van Den Bosch et al., 2006). However, there are several lines of evidence ⁎ Corresponding author at: Department of Neurology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. Fax: + 81 43 226 2160. E-mail address: [email protected] (S. Kuwabara). 0014-4886/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2011.08.015
that the predominant anatomical sites of origin of fasciculations are the distal motor axons (Layzer, 1994), although some fasciculations arise proximally early in the disease (de Carvalho and Swash, 1998). Therefore, most fasciculations arise from axons rather than neuronal cell bodies, suggesting altered membrane properties in motor axons of ALS. Previous axonal excitability studies in ALS patients have shown two types of ion channel abnormalities, reduced fast potassium currents and increased persistent sodium currents, both of which lead to axonal hyperexcitability (Bostock et al., 1995; Kanai et al., 2006; Mogyoros et al., 1998; Vucic and Kiernan, 2006). However, excitability indices measured by the neurophysiological technique are indirect measures of axonal ion channel function, and so far direct morphological examinations of altered ion channel expression have never been performed. By using immunohistochemistry, we studied the changes in expression of the ion channels in motor axons obtained from five autopsy cases of sporadic ALS. Materials and methods ALS patients and control subjects We studied post-mortem materials obtained from five patients (3 men and 2 women) with pathologically-proven ALS. Clinical profiles are shown in Table 1. The median survival time was 33 months (range, 22 to 49 months). None of them had a family history of ALS.
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Table 1 Clinical profiles of ALS patients and control subjects. Subject
Age at death gender
Site of onset
Survival time (months)
Postmortem delay (h)
Bulbar Bulbar Upper limb Upper limb Upper limb
49 22 25 27 42
2 5 4 6 2
96 84
4 12
Patient 1 2 3 4 5
ALS ALS ALS ALS ALS
64 73 64 70 68
Control 1 2
LE AD
57 F 84 M
M F M F M
ALS= amyotrophic lateral sclerosis; LE= leukoencepharopathy; AD= Alzheimer's disease.
TDP-43 immunostaining in the cytoplasm of spinal motor neurons was positive in all five patients. Two patients with Alzheimer disease or leukoencephalopathy with spheroids served as disease controls (Freeman et al., 2009). The research protocol was approved by the local Research Ethics Committees. Informed consent of immunohistological investigations was obtained their family members. Immunohistochemistry In all subjects, the hypoglossal nerves, and C7 and L5 ventral/dorsal roots were collected 2–12 h after death (Table 1). C7 and L5 roots were chosen because of the anatomical reason; these roots are located in the center of the cervical (C5–T1) and lumbar (L3–S1) enlargements of the spinal cord. Tissues were post-fixed for 30 min in 4% paraformaldehyde in 0.1 M PB, pH 7.4. And these were cryoprotected with 30% sucrose in phosphate-buffered saline (PBS), pH 7.4, overnight at 4 °C, as described previously (Ishibashi et al., 2004). After flat embedding in rectangular moulds in OCT medium, nerves were sectioned longitudinally at 8 μm. Cryosections were permeabilized for 1 h in 0.1 M PB, pH7.4, containing 0.3% Triton X-100 and 10% goat serum (PBTGS). For double-labeling experiments, sections were incubated overnight at 4 °C with primary antibodies diluted to appropriate concentrations in PBTGS. Primary antibodies were a rabbit polyclonal antibody against Kv1.2 channel (1:200) and mouse monoclonal antibody against pan sodium channel (1:500). These were purchased from Alomone labs (Jerusalem, Israel) and Sigma Co. (St. Louis, Missouri, USA). The sections were thoroughly rinsed in PBS, followed by application of fluorescently labeled secondary antibodies used were Alexa Fluor 488-labeled goat anti-rabbit immunoglobulin G (IgG) and Alexa Fluor 594-labeled goat anti-mouse IgG (1:2000; Molecular Probes, Eugene, Oregon, USA). Analysis of tissue sections For analysis of nerve sections, multiple images were captured with confocal microscope FV10i (Olympus Co Ltd, Tokyo, Japan). In each nerve, 30 fields were randomly captured and each fieldconsisted of 117.85 × 117.85 μm. To ensure random sampling, 10 fields were selected for analysis as performed in previous studies (Craner et al., 2003). Sensitivities to the both fluorescence were fixed for capturing each images. Signal intensities of the immunostaining signal at sodium and potassium channel cluster was obtained by manual outlining of each channels, using Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA) integrated densitometry function to calculate mean signal intensities for the outline area. (0–255) Background signal was subtracted from both ALS and control images (Craner et al., 2003). TDP-43 histochemistry In post-mortem materials from 5 ALS patients, sections (10 mm) of formalin-fixed, paraffin-embedded spinal cord were examined. Sections
were immunostained with anti phosphorylated TDP-43 antibodies (mouse monoclonal, 1:5000; rabbit polyclonal, 1:5000; Cosmobio, Tokyo, Japan).
Multiple Excitability Measurements Multiple excitability properties were measured for the median nerve at the wrist in Patient 5, (QTRAC version 4.3 with multiple excitability protocol TRONDXM2; copyright, Prof. Hugh Bostock, Institute of Neurology, London, UK), as reported elsewhere (Kanai et al., 2006; Kiernan et al., 2000). Briefly, in the threshold electrotonus studies, the membrane potential was altered by use of DC polarizing currents that were 40% of the unconditioned threshold. Depolarizing and hyperpolarizing currents were used, each lasting 100 ms, and their effects on the threshold current for the test motor responses were examined. The recovery cycle of axonal excitability after a single supramaximal stimulus was measured by delivering the test stimulus at different intervals after the conditioning stimulus. The interval between the conditioning and test stimulation were changed systematically from 2 to 200 ms.
Statistical analyses A repeated measurement analysis (Fitzmaurice et al., 2004) was performed to assess the immunostaining intensity of potassium and sodium channel clusters. F-test was used for adjustment of comparison between controls and ALS patients. Dunnett's multiple comparison tests were also applied between controls and ALS subgroups. In all comparisons, p-value of less than 0.05 was considered to be statistically significant. All statistical analyses were performed by the SAS software program, version 9.2 (SAS Institute Inc., Cary, NC, USA).
Results Immunostaining of potassium channel clusters In ALS patients, the immunoreactivity of potassium channel clusters (Kv1.2 channels) was prominently reduced in the ventral roots at both the C7 and L5 levels. Fig. 1 shows examples of immunostaining in the C7 roots of Patient 3 and a control subject with Alzheimer disease. In the ALS patient, the immunoreactivity of Kv1.2 channels was nearly lost in the C7 ventral root (Fig. 1C), but the potassium channel clusters were similarly stained in the ALS dorsal root and control ventral/dorsal roots. Table 2 shows results of the immunostaining intensity measured by densitometry for all subjects. For potassium channel clusters, the mean intensities markedly decreased in the C7 and L5 ventral roots in the ALS group than in the controls. In the hypoglossal nerves, the intensities were reduced in patients with bulbar onset (patients 1 and 2), but the difference did not reach a significance because of severe loss of axons and thereby a small number of nodes examined. Patient 3 with upper limb onset showed the decreased intensities predominantly in the C7 ventral root with relatively preserved those in the L5 ventral root (Fig. 2). ALS patients with lower limb onset (patients 4 and 5) had prominently reduced intensities in both the C7 and L5 ventral roots, whereas the extent of reduction was greater in the L5 roots. The immunostaining intensities in the C7 and L5 dorsal roots were similar for ALS patients and control subjects.
Immunostaining intensity of sodium channel clusters Table 2 also shows the immunostaining intensities of nodal sodium channel clusters. The intensities were not significantly different in the hypoglossal nerve, C7 ventral and dorsal roots, and L5 ventral and dorsal roots of control subjects and ALS patients.
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Discussion Our results show that in ALS patients, expression of Kv1.2 channels is markedly reduced in motor axons, and normal in sensory axons in both the C7 and L5 spinal roots levels, indicating selective loss of potassium channels in motor axons of ALS patients. These findings provide the morphological and biological basis for the previous neurophysiological data of reduced accommodative axonal potassium currents, and resulting increased axonal excitability in ALS motor axons (Bostock et al., 1995; Kanai et al., 2006; Vucic and Kiernan, 2006).
Prominently reduced expression of potassium channel in the ventral roots
Fig. 1. Immunohistochemistry of sodium and potassium channels in a patient with ALS and control subject. Longitudinal sections of the C7 ventral/dorsal roots double immunostained for pan-Nav channel (red) and Kv1.2 (green) in a post-mortem specimen obtained from autopsy cases of Alzheimer disease (A, B) and amyotrophic lateral sclerosis (C, D) (ALS; Patient 3 in Table 1). Note marked loss of the immunoreactivity for Kv1.2 channels in ALS ventral root (C). A scale bar indicates 10 μm.
Axonal excitability studies Patient 5 underwent axonal excitability studies in median motor axons at the wrist 22 months before death. Fig. 3 shows threshold electrotonus and the recovery cycle of axonal excitability after a single supramaximal conditioning stimulus. Threshold electrotonus demonstrated greater threshold changes in the early depolarizing phase (less accommodative currents mediated by fast potassium channels), and the recovery cycle showed increased superexcitability (less fast potassium currents). These findings suggested reduced fast potassium currents (Kv1.2 channels) as reported in previous studies (Bostock et al., 1995; Kanai et al., 2006; Vucic and Kiernan, 2006).
Our findings are highly consistent with previous neurophysiological study results. Axonal excitability studies in ALS patients have shown decreased fast and slow potassium currents in motor axons of the median or ulnar nerve at the wrist (Bostock et al., 1995; Kanai et al., 2006; Vucic and Kiernan, 2006). The functional reduction of potassium currents is now proved to be caused by the decreased channel expression. A microarray analysis in the spinal motor neurons of ALS patients has shown reduced mRNA expression of several types of potassium channels (KCNA1, KCNA2 and KCNQ2) (Jiang et al., 2005). Kv1.1 is a shaker related voltage-gated potassium channel in humans encoded by the KCNA1 gene, Kv1.2 channel examined in this study is encoded by the KCNA2 gene, and Kv7.1 channel is encoded by the KCNQ2 gene. Combined with the microarray study results, our findings suggest that the reduced mRNA levels of potassium channels in the spinal motor neurons lead to less expression of potassium channel proteins in peripheral motor axons in ALS. In addition, previous studies in transgenic mice revealed that axonal transport deficiencies could play important roles in the pathophysiology of ALS, and impaired transport of axonal membrane proteins including several potassium channels or proteins required for maintaining potassium channels might also contribute to a reduction of expression in potassium channels (LaMonte et al., 2002). Overall, the combination of decreased potassium channel mRNA and impaired axonal transport could result in markedly reduced axonal potassium channel expression, and thereby decreased potassium currents. Because potassium currents are compensatory outward rectifying conductances in response to depolarization, their decrease would results in substantial increases in axonal excitability and spontaneous firing of motor axons (e.g., fasciculations). It is recently suggested that a subset of ALS patients may have serum anti-potassium channel antibodies (Nwosu et al., 2010; Thompson and
Table 2 The immunostaining intensity of potassium and sodium channel clusters. Control (n = 2) LS mean (95%CI)
Amyotrophic lateral sclerosis All (n = 5)
Bulbar (n = 2)
Upper limb (n = 1)
Lower limb (n = 2)
LS mean (95%CI)
P-valuea
LS mean (95%CI)
P-valueb
LS mean (95%CI)
P-valueb
LS mean (95%CI)
P-valueb
Potassium channel cluster Hypoglossal nerve C7 ventral root C7 dorsal root L5 ventral root L5 dorsal root
28.4 45.6 42.2 47.1 46.6
(− 45.8–102) (32.6–58.5) (29.3–55.0) (30.7–63.4) (36.8–56.3)
18.9 18.7 34.8 24.1 42.1
(− 0.71–38.5) (9.27–28.2) (29.7–40.0) (13.6–34.6) (37.1–47.1)
0.55 0.0061 0.10 0.024 0.30
5.77 23.7 37.4 42.7 44.4
(− 74.3–85.8) (1.39–46.0) (21.9–52.9) (21.5–63.9) (39.5–49.2)
0.73 0.16 0.81 0.92 0.84
21.7 24.0 33.9 41.6 36.1
(− 55.4–98.9) (3.95–44.1) (19.0–48.8) (17.9–65.4) (23.5–48.8)
0.98 0.14 0.51 0.88 0.24
21.6 12.6 34.3 9.73 42.8
(− 32.8–76.1) (− 3.73–29.0) (20.5–48.1) (− 6.26–25.7) (33.1–52.6)
0.97 0.034 0.51 0.030 0.73
Sodium channel cluster Hypoglossal nerve C7 ventral root C7 dorsal root L5 ventral root L5 dorsal root
34.7 48.7 52.6 54.3 58.9
(2.39–67.0) (6.40–91.0) (15.9–89.3) (− 2.11–110) (29.9–87.9)
31.9 46.2 45.6 68.0 57.3
(22.6–41.3) (25.0–64.4) (30.8–60.5) (55.6–80.3) (45.0–69.5)
0.70 0.87 0.54 0.37 0.86
31.5 40.8 43.5 74.9 60.1
(− 7.34–70.4) (− 4.27–85.9) (6.38–80.8) (16.8–133) (31.2–88.9)
0.98 0.95 0.90 0.78 0.99
36.3 66.5 44.8 74.6 50.6
(2.12–70.5) (6.41–126) (− 6.70–96.3) (− 5.39 -154) (9.67–91.5)
0.99 0.80 0.95 0.85 0.91
29.9 40.5 48.1 58.3 57.7
(5.83–54.1) (− 2.50–83.6) (11.2–85.0) (1.74–114) (28.5–86.9)
0.91 0.94 0.98 0.99 0.99
LS Mean: least square mean. a F test P-value by repeated ANOVA. b Adjusted P-value by multiple tests using Dunnett's test.
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Fig. 2. Immunostaining reactivity measured by densitometry. The immunostaining reactivity of potassium channel (Kv1.2 channel) cluster measured by densitometry in the C7 and L5 ventral/dorsal roots obtained from autopsy cases of amyotrophic lateral sclerosis (ALS; n = 5) and controls (n = 2). Note prominent reduction of the intensities in the ALS ventral roots. In the dorsal roots, the immunostaining intensity was preserved in ALS patients. B = Bulbar onset (n = 2); U = upper limb onset (n = 1); L = lower limb onset (n = 2). *p b 0.05; **p b 0.001.
Gan, 2011). We did not measure the antibodies in our patients, but this issue should be clarified in future studies. In this study, immunostaining intensities of potassium channel clusters in sensory axons were preserved in ALS. Clinically and electrophysiologically, sensory axons are preserved in ALS (de Carvalho, 2000; Ertekin, 1967; Fincham and Vanallen, 1964), and excitability of sensory axons is not altered in ALS (Mogyoros et al., 1998). It is generally accepted that sensory axons are not involved in ALS, and our results indicate altered expression of potassium channels is selective for motor axons in ALS. Distribution of the reduced potassium channels and clinical phenotypes Our results show a tendency that the immunostaining intensities of potassium channel cluster were more severely decreased in the spinal roots corresponding to the site of symptom onset than other segments (Table 2, Fig. 2); ALS patients with lower limb onset tended to show the lower immunostaining intensity in the lumbar roots. These findings appear to be consistent with a pattern of progression in the ALS pathology (Brooks, 1996; Caroscio et al., 1987). Lower motor neuron degeneration in ALS is a focal process that advances contiguously (Ravits et al., 2007). Reduction of potassium currents leads to hyperexcitability in motor neurons/axons, and motoneuron may no longer meet the demands of its ATP-dependent processes, and ultimately dies (Henneberry et al., 1989; Novelli et al., 1988; Van Den Bosch et al., 2006). Conversely, the predominancy of potassium channel changes may determine the site of onset in ALS. Preserved immunostaining intensities of nodal sodium channels Our findings also show that there is no significant change in sodium channel expression in motor axon of ALS patients. Previous excitability studies revealed an increase in persistent sodium current in motor axon with ALS (Bostock et al., 1995; Kanai et al., 2006;
Mogyoros et al., 1998; Vucic and Kiernan, 2006). Sodium channels in the nodes of Ranvier of the peripheral nerves consist of only Nav1.6 channels, but there are two functionally different channels among Nav1.6 channels; in normal mammalian myelinated nerves, 1.0%–2.5% of the total nodal sodium channels are open at rest, and therefore termed “persistent” channels (Mogyoros et al., 1998). Our study failed to confirm changes in sodium channels. Immunohistochemistry cannot differentiate classical transient sodium channels and persistent channels, because these channels are structurally the same. Therefore normal sodium channel expression in this study does not exclude changes in persistent sodium currents. Another possible reason for the lack of changes in sodium channels in ALS includes the disease stage at the time of examination. Our previous study has shown that persistent sodium currents estimated by strength-duration time constant were increased in the whole ALS group, but were not significantly different in the end stage when patients develop severe muscle wasting (Kanai et al., 2006). In this regard, our findings were consistent with previous studies that suggest increase persistent sodium channels in the whole ALS patients examined (Mogyoros et al., 1998). Relevance to treatment Our morphological study and previous physiological studies revealed reduction of potassium channel expression and resulting decreased potassium currents in motor axon with ALS (Bostock et al., 1995; Kanai et al., 2006; Mogyoros et al., 1998). Investigating ionic mechanisms is of clinical relevance, because once a specific ion channel abnormality is identified, its pharmacologic modulation would provide a new therapeutic option. Riluzole is the only therapeutic agent for which effectiveness in human ALS patients has been confirmed by a large randomized placebo-controlled trial (Lacomblez et al., 1996). The positive effects of riluzole are related with its anti-excitotoxic properties inhibition of
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References
Fig. 3. Excitability testing in Patient 1 before death. Multiple axonal excitability testing for the median nerve at the wrist in a patient with amyotrophic lateral sclerosis (ALS; Patient. 5 in Table 1) 22 months before death. Thick lines indicate data from an ALS patient, and thin lines, the mean curves of age-matched normal subjects (n = 10). A. Threshold electrotonus showed greater threshold changes in the early depolarizing phase (arrow). B. Recovery cycle of axonal excitability after a single supramaximal conditioning stimulus showed greater supernormality in an ALS patient (arrow). These finding suggest reduced fast potassium currents mediated by Kv1.2. Strength-duration time constant was 0.43 ms (normal mean + − SEM; 0.43 + − 0.02 ms; n = 10).
glutamate release, blocking of voltage-dependent sodium channels, and activation of potassium channels (Mantz et al., 1992; Mathie and Veale, 2007; Song et al., 1997). Such effects could lead to neuroprotection and slowing of disease progression in ALS. However the clinical effects of riluzole are not entirely sufficient. Whereas many clinical trials for ALS have failed (Aggarwal and Cudkowicz, 2008), new therapeutic strategies for ALS are definitely necessary. Our results raise the possibility that modulation of axonal inonic currents is novel treatments for ALS. Particularly, potassium channel openers, such as retigabine, possibly could increase accommodative potassium currents, and therefore decrease axonal excitability in ALS patients. At present, it is unclear whether the altered ionic channels are the cause or result of the disease, but we believe continuous and extensive fasciculations would increase metabolic demands or oxidative stress in motor neurons, and enhance motor neuronal death. Acknowledgments This work was partly supported by Grants-in-Aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health, Labour and Welfare of Japan (S.K.).
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